The primary transcripts of eukaryotic structural genes (precursor mRNAs; pre-mRNAs) contain intervening sequences (introns) that are removed by RNA splicing. In some instances, alternative splicing of a common pre-mRNA provides an important mechanism to regulate gene expression. Pre-mRNA splicing occurs in a ribonucleoprotein (RNP) complex called the spliceosome, which is composed of a large number of proteins and multiple U small nuclear RNP particles (U snRNPs). A variety of mammalian protein splicing factors contain an arginine-serine rich (RS) domain required to promote splicing. During the past budget period we have shown that direct contact with the branchpoint and 5' splice site is a general mechanism by which RS domains promote spliceosome assembly and splicing. Our studies have revealed a pathway of sequential interactions between RS domains and splicing signals during mammalian spliceosome assembly. Experiments are proposed to understand how RS domains are directed to splicing signals, the basis by which RS domain-splicing signal-interactions promote spliceosome assembly and splicing, and the generality of the proposed mechanism. The U2 snRNP Auxiliary Factor (U2AF) is an essential splicing factor that binds to the polypyrimidine (Py) tract/3' splice site and initiates spliceosome assembly. hUAP56, a member of the DExD/H box family of RNA-dependent ATPases interacts with the large U2AF subunit (U2AF65). Both U2AF and UAP56 were originally identified in our laboratory and we will continue to study how these proteins function to promote spliceosome assembly and splicing. Genome sequencing and expression studies have revealed the existence of proteins that are highly related to the small U2AF subunit, U2AF35. We will use genetic, biochemical and molecular biological approaches to study the function of U2AF35-related proteins and identify pre-mRNAs upon which they act. Our studies on RS domains led us to develop a powerful RNA/protein crosslinking procedure that can be used to determine whether specific domains of proteins involved in RNA processing directly contact discrete pre-mRNA regions. We will use this experimental approach to study the mechanism of action of other splicing factors and regulators. [unreadable] [unreadable] [unreadable]